Construction Of A 3D Printed Magnesium Phosphate/Polycaprolactone Scaffold For In Situ Tissue Regeneration And Its Osteogenesis In Vivo And In Vitro Experimental Study

Objective:The integrity of the oral and maxillofacial bone structure has an extremely important influence on completing the functions such as normal chewing,speech,and presenting the aesthetic features of the face.In situ tissue regeneration is widely adopted in the field of bone and cartilage tissue repair.The scaffold is implanted directly into the defect site of the body.the use of the inherent biological activity and differentiation ability of scaffold materials to recruit the migration,proliferation and differentiation of cells in vivo to achieve the repair of defective tissue.In order to achieve the function of in situ tissue engineering scaffold,the most commonly adopted strategy is to add bio-responsive molecules to the scaffold materials,and tissue healing and regeneration can be achieved by the stimulation of bio-responsive molecules.At present,the hot research on scaffold materials is mainly focused on how to combine different biomaterials to prepare high-performance and rapid prototyping composites.As a result,the new materials can overcome the defects of previous materials.It has a better performance to adapt to the micro-environment in the body,and plays a better role in repairing the damaged parts.PCL scaffold has been widely used in bone tissue engineering,it has good biocompatibility,degradability,processability and mechanical properties,but simple PCL scaffold also has some disadvantages such as poor biological activity,poor hydrophilic ability and weak osteogenesis induction,these problems limit its application in the biomedical field.3D printed bone repair stents are able to customize stents of different appearance sizes according to patient needs to meet the needs of different defect locations.Biodegradable polymer scaffolds prepared by FDM bioprinting in 3D bioprinting methods can serve as bone tissue engineering scaffolds,but have disadvantages such as smooth surface and hindering effective cell attachment,which need to be improved.In this study,Mg₃（PO₄）₂was fused and mixed with PCL to prepare in situ tissue engineering scaffolds without adding seed cells using FDM bioprinting.Different ratios of Mg₃（PO₄）₂/PCL scaffold materials were prepared,physical and chemical characterization,mechanical properties and degradation properties were analyzed,to study the influence of scaffold structure and Mg₃（PO₄）₂content on scaffold properties.Through the in vitro and in vivo experiments of rat tibial bone defect model,the biocompatibility and bone defect repair performance of the new scaffold material were analyzed.The Mg₃（PO₄）₂/PCL scaffold material with the best osteogenic performance ratio was selected to verify the osteogenic performance of the scaffold in the rabbit mandibular bone defect model.Methods:1.Mg₃（PO₄）₂and PCL were mixed at ratio of 5:95,10:90 and 20:80（W/W）as experimental group,and PCL scaffolds as blank control group,printing with FDM biological printer.2.The morphology of four groups of scaffolds was observed by SEM,the pore structure and micro-morphology of four groups of scaffolds was obtained,and the surface area ratio of Mg₃（PO₄）₂on the scaffolds was calculated by Imge J（1.48V）software.3.The hydrophilicity of scaffolds was determined by contact angle test,The contact angles of the four scaffolds of PCL,PCL#5MgP,PCL#10MgP and PCL#20MgP were 122.55±5.35,113.86±2.53,106.6±7.77 and 95.54±3.20,respectively.4.The composition of different groups of scaffolds was determined by Fourier transform infrared spectroscopy（FITR）,Thermogravimetric analysis（TGA）and X-ray diffraction（XRD）.5.The shear viscosity and mechanical properties of scaffolds were measured.6.The degradation performance and the release profile of Mg²⁺from the four groups of scaffolds were examined.7.The PH values of the four groups were determined.8.MC3T3-E1 cells and scaffolds were inoculated and cultured in a three-dimensional culture device,and the cell adhesion and proliferation capacity of the scaffolds was evaluated by live-dead cells staining,CCK-8 experiments,and scanning electron microscopy.9.The scaffolds were immersed in simulated body fluid for 30 days,and SEM observed the scaffolds surface morphology and performed EDS element scanning analysis of Ca elements to evaluate the mineralization ability of different groups of scaffolds.10.MC3T3-E1 cells were planted on different scaffolds using a three-dimensional cell culture device.and the osteogenic ability in vitro of the scaffold was evaluated by ALP staining,ALP activity detection as well as ARS staining kit.11.The rat tibial defect model was established and four groups of scaffolds were implanted into the rat tibial defect model,Besides,the biocompatibility and osteogenic properties in vivo were detected by imaging and histology.12.The rabbit mandibular defect model was established,Mg₃（PO₄）₂/PCL scaffold material with optimal osteogenic performance ratio was selected as the experimental group,PCL scaffold was used as the control group,and a blank control group was set up.Osteogenic performance was detected by imaging 8 weeks after scaffold implantation.13.Statistical analysis of experimental data.Results:1.The Micro-CT 3D reconstruction image of the scaffold showed a regular porous structure with uniform fiber and pore size,SEM images showed that Mg₃（PO₄）₂particles were uniformly distributed in the scaffold,the fiber diameter of the scaffold was between 330.45±25.19μm,the scaffold showed a porous structure,the horizontal hole e\was square,the side length was 410.83±18.38μm,and the interlayer pore of the scaffold was rectangular with a height of 89.32±3.28μm.2.With the increase of Mg₃（PO₄）₂content,the contact angle decreases and the scaffold changed from hydrophobic surface to hydrophilic surface.3.The study results of FTIR,XRD and TGA showed that Mg₃（PO₄）₂was successfully mixed into the scaffold,and the shear viscosity of the scaffold increases with the increase of Mg₃（PO₄）₂content.4.The gradual increase of Mg₃（PO₄）₂content can significantly increase the compressive strength of the scaffold,but reduce the tensile strength.5.The scaffold can slowly degrade and gradually release Mg²⁺,the pH value of the scaffold was between 7.25 and 7.5.6.The adhesion and proliferation experiments of cells on the scaffold showed the non-toxicity of the Mg₃（PO₄）₂/PCL scaffold.Compared with the control group,the experimental group has enhanced cell adhesion ability,good uniform cell distribution and active cell proliferation.7.The pore structure of the scaffold and the addition of Mg₃（PO₄）₂enhanced the mineralization ability of the scaffold.8.The addition of Mg₃（PO₄）₂in the experimental group could improve the ALP activity and calcium deposition capacity of MC3T3-E1cells,and the PCL#20MgP scaffold group had the best bone differentiation performance in vitro.9.The results of imaging and histological examination showed that the osteogenic effect increased gradually when Mg₃（PO₄）₂was added to the scaffold in the rat tibial bone defect model experiment.Besides,the higher the content of Mg₃（PO₄）₂was,the more obvious the osteogenic enhancement effect was.The osteogenic effect of PCL#20MgP scaffold group was the most obvious,which was coincident with the results of in vitro experiment.10.In the rabbit mandibular defect model experiment,it was further confirmed that the repair effect of PCL#20MgP scaffold group on rabbit maxillofacial bone defect was significantly better than that of PCL group and blank control group.Conclusion:1.In this study,the new Mg₃（PO₄）₂/PCL in situ tissue repair scaffold material was successfully prepared using FDM biological 3D printing technology.The scaffold has good pore structure.With the increase of Mg₃（PO₄）₂content in the scaffold,the compressive strength of the scaffold increases,and the hydrophilic enhances and pH values are stable.The scaffold can slowly degrade and release Mg²⁺.2.In vitro experiments showed that the pore structure and Mg₃（PO₄）₂promoted the cell adhesion,proliferation,mineralization and bone differentiation properties of the PCL scaffold.In vivo osteogenesis experiments in the rat tibial bone defect model also confirmed that bone differentiation performance of the PCL#20MgP scaffolds were optimal,which was consistent with the in vitro results.3.The results showed that PCL#20MgP has good osteogenic properties in mandibular bone defect repair,and it can be used as a new material for maxillofacial bone defect repair.